Mammalian cells respond to extracellular stimuli by activating signaling cascades that are mediated by members of the mitogen-activated protein (MAP) kinase family, which include the extracellular signal regulated kinases (ERKs), the p38 MAP kinases and the c-Jun N-terminal kinases (JNKs). MAP kinases (MAPKs) are activated by a variety of signals including growth factors, cytokines, UV radiation, and stress-inducing agents. MAPKs are serine/threonine kinases and their activation occurs by dual phosphorylation of threonine and tyrosine at the Thr-X-Tyr segment in the activation loop. MAPKs phosphorylate various substrates including transcription factors, which in turn regulate the expression of specific sets of genes and thus mediate a specific response to the stimulus.
One particularly interesting kinase family are the c-Jun NH2-terminal protein kinases, also known as JNKs. Three distinct genes, JNK1, JNK2, JNK3 have been identified and at least ten different splicing isoforms of JNKs exist in mammalian cells [Gupta et al., EMBO J., 15:2760-70 (1996)]. Members of the JNK family are activated by proinflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β), as well as by environmental stress, including anisomycin, UV irradiation, hypoxia, and osmotic shock [Minden et al., Biochemica et Biophysica Acta, 1333:F85-F104 (1997)].
The down-stream substrates of JNKs include transcription factors c-Jun, ATF-2, Elk1, p53 and a cell death domain protein (DENN) [Zhang et al. Proc. Natl. Acad. Sci. USA, 95:2586-91 (1998)]. Each JNK isoform binds to these substrates with different affinities, suggesting a regulation of signaling pathways by substrate specificity of different JNKs in vivo [Gupta et al., supra].
JNKs, along with other MAPKs, have been implicated in having a role in mediating cellular response to cancer, thrombin-induced platelet aggregation, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis and heart disease. The therapeutic targets related to activation of the JNK pathway include chronic myelogenous leukemia (CML), rheumatoid arthritis, asthma, osteoarthritis, ischemia, cancer and neurodegenerative diseases.
Several reports have detailed the importance of JNK activation associated with liver disease or episodes of hepatic ischemia [Nat. Genet. 21:326-9 (1999); FEBS Lett. 420:201-4 (1997); J. Clin. Invest. 102:1942-50 (1998); and Hepatology 28:1022-30 (1998)]. Therefore, inhibitors of JNK may be useful to treat various hepatic disorders.
A role for JNK in cardiovascular disease such as myocardial infarction or congestive heart failure has also been reported as it has been shown that JNK mediates hypertrophic responses to various forms of cardiac stress [Circ. Res. 83:167-78 (1998); Circulation 97:1731-7 (1998); J. Biol. Chem. 272:28050-6 (1997); Circ. Res. 79:162-73 (1996); Circ. Res. 78:947-53 (1996); and J. Clin. Invest. 97:508-14 (1996)].
It has also been demonstrated that the JNK cascade plays a role in T-cell activation, including activation of the IL-2 promoter. Thus, inhibitors of JNK may have therapeutic value in altering pathologic immune responses [J. Immunol. 162:3176-87 (1999); Eur. J. Immunol. 28:3867-77 (1998); J. Exp. Med. 186:941-53 (1997); and Eur. J. Immunol. 26:989-94 (1996)].
A role for JNK activation in various cancers has also been established, suggesting the potential use of JNK inhibitors in cancer. For example, constitutively activated JNK is associated with HTLV-1 mediated tumorigenesis [Oncogene 13:135-42 (1996)]. JNK may play a role in Kaposi's sarcoma (KS) because it is thought that the proliferative effects of bFGF and OSM on KS cells are mediated by their activation of the JNK signaling pathway [J. Clin. Invest. 99:1798-804 (1997)]. Other proliferative effects of other cytokines implicated in KS proliferation, such as vascular endothelial growth factor (VEGF), IL-6 and TNFα, may also be mediated by JNK. In addition, regulation of the c-jun gene in p210 BCR-ABL transformed cells corresponds with activity of JNK, suggesting a role for JNK inhibitors in the treatment for chronic myelogenous leukemia (CML) [Blood 92:2450-60 (1998)].
JNK1 and JNK2 are widely expressed in a variety of tissues. In contrast, JNK3 is selectively expressed in the brain and to a lesser extent in the heart and testis [Gupta et al., supra; Mohit et al., Neuron 14:67-78 (1995); and Martin et al., Brain Res. Mol. Brain Res. 35:47-57 (1996)]. JNK3 has been linked to neuronal apoptosis induced by kainic acid, indicating a role of JNK in the pathogenesis of glutamate neurotoxicity. In the adult human brain, JNK3 expression is localized to a subpopulation of pyramidal neurons in the CA1, CA4 and subiculum regions of the hippocampus and layers 3 and 5 of the neocortex [Mohit et al., supra]. The CA1 neurons of patients with acute hypoxia showed strong nuclear JNK3-immunoreactivity compared to minimal, diffuse cytoplasmic staining of the hippocampal neurons from brain tissues of normal patients [Zhang et al., supra]. Thus, JNK3 appears to be involved in hypoxic and ischemic damage of CA1 neurons in the hippocampus.
In addition, JNK3 co-localizes immunochemically with neurons vulnerable in Alzheimer's disease [Mohit et al., supra]. Disruption of the JNK3 gene caused resistance of mice to the excitotoxic glutamate receptor agonist kainic acid, including the effects on seizure activity, AP-1 transcriptional activity and apoptosis of hippocampal neurons, indicating that the JNK3 signaling pathway is a critical component in the pathogenesis of glutamate neurotoxicity [Yang et al., Nature, 389:865-870 (1997)].
Based on these findings, JNK signaling, especially that of JNK3, has been implicated in the areas of apoptosis-driven neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, ALS (amyotrophic lateral sclerosis), epilepsy and seizures, Huntington's disease, traumatic brain injuries, as well as ischemic and hemorrhaging stroke.
The Src-family of kinases are implicated in cancer, immune system dysfunction, and bone remodeling diseases. For general reviews, see Thomas and Brugge, Annu. Rev. Cell Dev. Biol. 13:513 (1997); Lawrence and Niu, Pharmacol. Ther. 77:81 (1998); Tatosyan and Mizenina, Biochemistry (Moscow) 65:49 (2000); and Boschelli et al., Drugs of the Future 2000, 25(7):717 (2000).
Members of the Src family include the following eight kinases in mammals: Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, Blk and Yrc. These are nonreceptor protein kinases that range in molecular mass from 52 to 62 kD. All are characterized by a common structural organization that is comprised of six distinct functional domains: Src homology domain 4 (SH4), a unique domain, SH3 domain, SH2 domain, a catalytic domain (SH1), and a C-terminal regulatory region [Tatosyan et al. Biochemistry (Moscow) 65:49-58 (2000)].
Based on published studies, Src kinases are considered as potential therapeutic targets for various human diseases. Mice that are deficient in Src develop osteopetrosis, or bone build-up, because of depressed bone resorption by osteoclasts. This suggests that osteoporosis resulting from abnormally high bone resorption can be treated by inhibiting Src [Soriano et al., Cell, 69:551 (1992) and Soriano et al., Cell, 64: 693 (1991)].
Suppression of arthritic bone destruction has been achieved by the overexpression of CSK in rheumatoid synoviocytes and osteoclasts [Takayanagi et al., J. Clin. Invest., 104:137 (1999)]. CSK, or C-terminal Src kinase, phosphorylates and thereby inhibits Src catalytic activity. This implies that Src inhibition may prevent joint destruction that is characteristic in patients suffering from rheumatoid arthritis [Boschelli et al., Drugs of the Future 2000, 25(7):717 (2000)].
Src also plays a role in the replication of hepatitis B virus. The virally encoded transcription factor HBx activates Src in a step required for propagation of the virus [Klein et al., EMBO J., 18:5019, (1999) and Klein et al., Mol. Cell. Biol., 17:6427 (1997)].
A number of studies have linked Src expression to cancers such as colon, breast, hepatic and pancreatic cancer, certain B-cell leukemias and lymphomas [Talamonti et al., J. Clin. Invest., 91:53 (1993); Lutz et al., Biochem. Biophys. Res. 243:503 (1998); Rosen et al., J. Biol. Chem., 261:13754 (1986); Bolen et al., Proc. Natl. Acad. Sci. USA, 84:2251 (1987); Masaki et al., Hepatology, 27:1257 (1998); Biscardi et al., Adv. Cancer Res., 76:61 (1999); Lynch et al., Leukemia, 7:1416 (1993)]. Furthermore, antisense Src expressed in ovarian and colon tumor cells has been shown to inhibit tumor growth [Wiener et al., Clin. Cancer Res., 5:2164 (1999) and Staley et al., Cell Growth Diff., 8:269 (1997)].
Other Src family kinases are also potential therapeutic targets. Lck plays a role in T-cell signaling. Mice that lack the Lck gene have a poor ability to develop thymocytes. The function of Lck as a positive activator of T-cell signaling suggests that Lck inhibitors may be useful for treating autoimmune disease such as rheumatoid arthritis [Molina et al., Nature, 357: 161 (1992)]. Hck, Fgr and Lyn have been identified as important mediators of integrin signaling in myeloid leukocytes [Lowell et al., J. Leukoc. Biol., 65:313 (1999)]. Inhibition of these kinase mediators may therefore be useful for treating inflammation [Boschelli et al., Drugs of the Future 2000, 25(7):717 (2000)].
The Aurora family of serine/threonine kinases is essential for cell proliferation [Bischoff, J. R. & Plowman, G. D. Trends in Cell Biology et al., 9:454-459 (1999); Giet et al. Journal of Cell Science, 112:3591-3601 (1999); Nigg Nat. Rev. Mol. Cell Biol. 2:21-32 (2001); Adams et al., Trends in Cell Biology 11:49-54 (2001)]. Inhibitors of the Aurora kinase family therefore have the potential to block growth of all tumour types.
The three known mammalian family members, Aurora-A (“1”), B (“2”) and C (“3”), are highly homologous proteins responsible for chromosome segregation, mitotic spindle function and cytokinesis. Aurora expression is low or undetectable in resting cells, with expression and activity peaking during the G2 and mitotic phases in cycling cells. In mammalian cells proposed substrates for Aurora include histone H3, a protein involved in chromosome condensation, and CENP-A, myosin II regulatory light chain, protein phosphatase 1,
Since its discovery in 1997 the mammalian Aurora kinase family has been closely linked to tumorigenesis. The most compelling evidence for this is that over-expression of Aurora-A transforms rodent fibroblasts [Bischoff et al., EMBO J., 17:3052-3065 (1998)]. Cells with elevated levels of this kinase contain multiple centrosomes and multipolar spindles, and rapidly become aneuploid. The oncogenic activity of Aurora kinases is likely to be linked to the generation of such genetic instability. Indeed, a correlation between amplification of the aurora-A locus and chromosomal instability in mammary and gastric tumours has been observed. [Miyoshi et al. Int. J. Cancer, 92:370-373 (2001) and Sakakura et al. British Journal of Cancer, 84:824-831 (2001)]. The Aurora kinases have been reported to be over-expressed in a wide range of human tumours. Elevated expression of Aurora-A has been detected in over 50% of colorectal [Bischoff et al., EMBO J., 17:3052-3065 (1998) and Takahashi et al., Jpn. J. Cancer Res., 91:1007-1014 (2000)] ovarian [Gritsko et al. Clinical Cancer Research, 9:1420-1426 (2003)], and gastric tumors [Sakakura et al., British Journal of Cancer, 84:824-831 (2001)], and in 94% of invasive duct adenocarcinomas of the breast [Tanaka et al. Cancer Research, 59:2041-2044 (1999)]. High levels of Aurora-A have also been reported in renal, cervical, neuroblastoma, melanoma, lymphoma, pancreatic and prostate tumour cell lines. [Bischoff et al., EMBO J., 17:3052-3065 (1998); Kimura et al. J. Biol. Chem., 274:7334-7340 (1999); Zhou et al., Nature Genetics, 20:189-193 (1998); Li et al., Clin Cancer Res. 9(3):991-7 (2003)]. Amplification/overexpression of Aurora-A is observed in human bladder cancers and amplification of Aurora-A is associated with aneuploidy and aggressive clinical behaviour [Sen et al., J Natl Cancer Inst., 94(17):1320-9 (2002)]. Moreover, amplification of the aurora-A locus (20q13) correlates with poor prognosis for patients with node-negative breast cancer [Isola, American Journal of Pathology 147, 905-911 (1995)]. Aurora-B is highly expressed in multiple human tumour cell lines, including leukemic cells [Katayama et al., Gene 244:1-7)]. Levels of this enzyme increase as a function of Duke's stage in primary colorectal cancers [Katayama et al., J. Natl Cancer Inst., 91:1160-1162 (1999)]. Aurora-C, which is normally only found in germ cells, is also over-expressed in a high percentage of primary colorectal cancers and in a variety of tumour cell lines including cervical adenocarinoma and breast carcinoma cells [Kimura et al., J. Biol. Chem. 274:7334-7340 (1999) and Takahashi et al., Jpn. J. Cancer Res., 91:1007-1014 (2000)].
Based on the known function of the Aurora kinases, inhibition of their activity should disrupt mitosis leading to cell cycle arrest. In vivo, an Aurora inhibitor therefore slows tumor growth and induces regression.
Elevated levels of all Aurora family members are observed in a wide variety of tumour cell lines. Aurora kinases are over-expressed in many human tumors and this is reported to be associated with chromosomal instability in mammary tumors.
Aurora-2 is highly expressed in multiple human tumor cell lines and levels increase as a function of Duke's stage in primary colorectal cancers [Katayama et al., J. Natl Cancer Inst., 91:1160-1162 (1999)]. Aurora-2 plays a role in controlling the accurate segregation of chromosomes during mitosis. Misregulation of the cell cycle can lead to cellular proliferation and other abnormalities. In human colon cancer tissue, the Aurora-2 protein has been found to be over expressed [Bischoff et al., EMBO J., 17: 3052-3065 (1998); Schumacher et al., J. Cell Biol., 143:1635-1646 (1998); Kimura et al., J. Biol. Chem., 272: 13766-13771 (1997)]. Aurora-2 is over-expressed in the majority of transformed cells. Bischoff et al found high levels of Aurora-2 in 96% of cell lines derived from lung, colon, renal, melanoma and breast tumors [Bischoff et al., EMBO J., 17:3052-3065 (1998)]. Two extensive studies show elevated Aurora-2 in 54% and 68% [Bishoff et al., EMBO J., 17:3052-3065 and Takahashi et al. Jpn J Cancer Res. 91:1007-1014 (2000)] of colorectal tumours and in 94% of invasive duct adenocarcinomas of the breast (Tanaka et al., Cancer Research, 59:2041-2044 (1999)].
Aurora-1 expression is elevated in cell lines derived from tumors of the colon, breast, lung, melanoma, kidney, ovary, pancreas, CNS, gastric tract and leukemias (Tatsuka et al 1998 58, 4811-4816).
High levels of Aurora-3 have been detected in several tumour cell lines, although it is restricted to testis in normal tissues [Kimura et al. Journal of Biological Chemistry, 274:7334-7340 (1999)]. Over-expression of Aurora-3 in a high percentage (c. 50%) of colorectal cancers has also been documented (Takahashi et al., Jpn J. Cancer Res. 91:1007-1014 (2001)]. In contrast, the Aurora family is expressed at a low level in the majority of normal tissues, the exceptions being tissues with a high proportion of dividing cells such as the thymus and testis [Bischoff et al EMBO J., 17:3052-3065 (1998)].
For further review of the role Aurora kinases play in proliferative disorders, see Bischoff et al., Trends in Cell Biology 9:454-459 (1999); Giet et al. Journal of Cell Science, 112:3591-3601 (1999); Nigg et al., Nat. Rev. Mol. Cell Biol., 2:21-32 (2001) et al., Trends in Cell Biology, 11:49-54 (2001); and Dutertre, et al. Oncogene, 21:6175-6183 (2002).
There is a continued need to develop potent inhibitors of JNKs, Src family kinases, and Aurora family kinases that are useful in treating or preventing various conditions associated with JNK, Src, and Aurora activation.